Identification of genetic determinants of bedaquiline resistance in Mycobacterium tuberculosis in Ural region, Russia

ABSTRACT Collecting data on rare Mycobacterium tuberculosis (Mtb) clinical isolates with resistance to the new anti-tuberculosis drug bedaquiline is an important task for improving antimicrobial susceptibility testing methods. Nanopore whole genome sequencing, the proportion method on Middlebrook 7H11 medium, and BACTEC MGIT 960 assays were used to analyze genotypic and phenotypic resistance to bedaquiline. We found four mutations: atpE I66M, atpE А63Р, Rv0678 А36Т, and Rv0678 S53P in five isolates with different levels of phenotypic bedaquiline resistance. IMPORTANCE Bedaquiline (BDQ) is a new anti-tuberculosis drug. The phenotypic and genotypic data describing the mechanism of drug resistance are critical for the design of rapid and accurate diagnostic tests. We consider that our work, which describes genotypic and phenotypic resistance to BDQ, can contribute to the standardization of drug susceptibility testing.

In this study, we investigated mutations associated with BDQ resistance in pheno typic-resistant Mtb isolates using whole genome sequencing (WGS) with Nanopore technology.

MATERIALS AND METHODS
During the period from November 2019 to December 2021, 239 Mtb isolates were obtained from various clinical samples from 203 TB patients who were treated at the Ural Research Institute for Phthisiopulmonology, a branch of the National Medical Research Center of Phthisiopulmonology and Infection Diseases.
Genomic DNA (gDNA) was extracted from aliquots of Mtb suspension prepared for DST using cetyltrimethylammonium bromide (CTAB) method (10).One nanogram of input gDNA was used for library preparation.For genomic DNA library preparation, all steps were performed according to the gDNA Ligation sequencing protocol-native barcoding (SQK-LSK109 with EXP-NBD04) (Oxford Nanopore Technologies, Oxford, UK).WGS was performed on MinION platform with R9 flow cells.Base calling was done using Guppy 6.2.1 basecaller.

RESULTS AND DISCUSSION
According to drug susceptibility testing results, 206 (86.2%) of the 239 isolates studied were MDR, including five (2.1%) with resistance to BDQ.WGS was performed on these five isolates, and the MIC of bedaquiline was determined by the proportion method on Middlebrook 7H11 medium, as well as using BACTEC MGIT 960 technology.
Sequencing on the Oxford Nanopore platform yielded median coverage depths ranging from 39 to 298.Mutations of the atpE gene were found in three isolates: I66M mutation was identified in two isolates with an MIC of 0.5 mg/L on Middlebrook 7H11 medium (resistant) and 4-8 mg/L on BACTEC MGIT 960 (resistant), an isolate with the А63Р mutation had an MIC of 2 mg/L on Middlebrook 7H11 medium (resistant) and 32 mg/L on BACTEC MGIT 960 (resistant) (Table 1).
According to catalog of mutation, atpE I66M and А63Р were determined as associ ated with resistance-interim (8).Computational analysis of the structural and functional  consequences of these variants revealed that resistance-associated variants are primarily localized to the drug-binding site, disrupting key interactions with BDQ and resulting in reduced binding affinity (15,16).Differences in binding affinity in these sites could be reflected in different MIC values.
Two more isolates had Rv0678 mutations: А36Т with an MIC of 0.25 mg/L on Middlebrook 7H11 medium (resistant) and 8 mg/L on BACTEC MGIT 960 (resistant).An isolate with S53P mutation had an MIC of less than 0.12 mg/L on Middlebrook 7H11 medium (sensitive) and 1 mg/L on BACTEC MGIT 960 (resistant).
Only Rv0678 S53P is marked as uncertain significance in catalog of mutation.Mutation Rv0678 А36Т is not listed in the catalog but previously was described as associated with a bedaquiline-resistant phenotype (low confidence) (17).It is impor tant to note that the Rv0678 S53P mutation was identified in Mtb isolates from BDQnaive patients, while the other patient had received BDQ for 13-36 months (Table 1).Resistance-conferring mutations in Rv0678 gene have also been previously reported in BDQ-naive MDR-TB patients (18)(19)(20).This finding highlights the importance of genotypic and phenotypic BDQ DST at baseline to ensure appropriate TB treatment.
No pepQ gene mutations were detected in all five isolates.

Conclusion
In this study, we analyzed genotypic and phenotypic resistance to BDQ in Mtb clinical isolates obtained from TB patients in Ural region, Russia.Our results contribute to the knowledge of the genetic basis of resistance to BDQ: mutation Rv0678 А36Т is not listed in the WHO catalog of mutation but previously was described as associated with a bedaquiline-resistant phenotype; Rv0678 S53P mutation was identified in Mtb isolates from BDQ-naive patient.We hope that these results can contribute to the standardiza tion of DST.

ETHICS APPROVAL
The study was approved by the Local Ethics Committee of the National Medical Research Center for Phthisiopulmonology and Infection Disease, Ekaterinburg, Russia (approval protocol no.102, 24.11.2021).Сlinical isolates included in this study were obtained from collections routinely assembled as a part of the practices of the microbiology laboratory.
No personal patients' data or experiments on humans or human tissues were utilized in this study.This study was retrospective and included only investigations of M. tuberculo sis clinical isolates, and neither the patient's diagnosis nor the treatment was changed.

TABLE 1
The genetic and phenotype data of Mycobacterium tuberculosis clinical isolates resistant to bedaquiline a